scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

TUMOR-ASSOCIATED FIBRINOLYSIS IN OVARIAN CARCINOMA - HPLC AND N-TERMINAL AMINO ACID ANALYSIS REVEAL THE PATHWAY OF DEGRADATION OF CROSSLINKED FIBRIN

1987 ◽  
Author(s):  
O Wilhelm ◽  
A Henschen ◽  
R Hafter ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
Ovarian Carcinoma ◽  
Ascitic Fluid ◽  
High Performance ◽  
Degradation Products ◽  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Reversed Phase ◽  
Fibrin Degradation Products ◽  
Sds Page

Crosslinked fibrin has been demonstrated by immunohistochemi-cal tests to occur around tumor plugs, on the surface and in the stroma of the tumor in ovarian cancer. High levels of D-Dimer (200-800μg/ml), the characteristic terminal degradation product of crosslinked fibrin, are found in ascitic fluid of patients with advanced ovarian cancer. These findings suggest that fibrin polymerisation and degradation are related to and even may influence tumor growth. The kind of proteases which are responsible for degradation of crosslinked fibrin is, however, unknown.lt was the aim of this study to evaluate whether plasmin and/or other proteases are involved in tumor-associated fibrinolysis. Therefore the total high-molecular-weight fibrin degradation products in ascitic fluid were purified by protamine sulfate precipitation, gel filtration, immunoadsorption and compared with the components of plasmin-degraded crosslinked fibrin, i.e. DD,DY,YX,DXD and DXY, by direct SDS-PAGE in the absence of mercaptoethanol and after excision of the bands, mercaptolysis and re-electrophoresis. Pronounced similarity between the two sets of fragments was observed. For further information the fragments from the two sources were mercaptolysed and their polypeptide chain components separated by reversed-phase high-performance liquid chromatography, the components being identified by N-terminal sequence analysis and SDS-PAGE. Highly similar patterns were obtained and components corresponding to γ-γ ,γ-γ1, β, β2 and α1 could be recognized. The findings provide strong evidence for plasmin being the primary protease involved in ovarian carcinoma-related fibrinolysis, (supported by Deutsche Forschungsgemeinschaft.SFB 207, A2).

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Extraction, Characterization and Antioxidant Activity in vitro of Proteins from Semen Allii Fistulosi

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3235
Author(s):  
Min Zuo ◽  
Xiao-xiao Liu ◽  
Di Liu ◽  
Hang-yun Zhao ◽  
Lu-lu Xuan ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
High Performance ◽  
Orthogonal Design ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Power ◽  
Raw Material ◽  
Extraction Solvent ◽  
Sds Page

Semen Allii Fistulosi (PSAF) is the seed of Allium fistulosum L. of the Liliaceae family. The purpose of this study was to extract, characterize, and evaluate the antioxidant activity in vitro of proteins. Using single factor and orthogonal design, the optimum conditions of extraction were determined to be as follows: extraction time 150 min, pH 8.5, temperature 60 °C, and ratio (v/w, mL/g) of extraction solvent to raw material 35. The isoelectric point of the pH was determined to be about 4.4 and 10.2, by measuring the protein content of PSAF solutions at different pH values. The amino acid composition of PSAF was determined by high performance liquid chromatography (HPLC), and the results suggested that the species of amino acids contained in the PSAF was complete. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed the molecular weight was mainly between 40 and 55 kDa, and Fourier-transform infrared spectroscopy (FTIR) characterized prevalent protein absorption peaks. PSAF exhibited potent scavenging activities against DPPH assays, via targeting of hydroxyl and superoxide radicals, while chelating Fe2+ activity and demonstrating weak reducing power. This work revealed that PSAF possessed potential antioxidant activity in vitro, suggesting potential for use of PSAF as a natural antioxidant.

Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea

1996 ◽  
Vol 42 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Solid State ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Performance Liquid Chromatographic ◽  
Exchange Chromatography ◽  
Glucosidase Activity ◽  
Apparent Homogeneity ◽  
Sds Page

The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.

scholarly journals Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit

2005 ◽  
Vol 187 (13) ◽  
pp. 4444-4450 ◽  
Author(s):  
Alaka Srivastava ◽  
Samuel I. Beale
Keyword(s):  
Molecular Mass ◽  
Column Chromatography ◽  
Aminolevulinic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Affinity Column ◽  
Chlorobium Vibrioforme ◽  
Sds Page

ABSTRACT δ-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His6 tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His6-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNAGlu and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of β-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

scholarly journals Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

1998 ◽  
Vol 64 (1) ◽  
pp. 62-67 ◽  
Author(s):  
Yukie Akutsu ◽  
Toshiaki Nakajima-Kambe ◽  
Nobuhiko Nomura ◽  
Tadaatsu Nakahara
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Water Soluble ◽  
Degrading Enzyme ◽  
Comamonas Acidovorans ◽  
Water Soluble Compound ◽  
Sds Page ◽  
Hydrophobic Adsorption

ABSTRACT A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2%N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45°C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity whenp-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Study on Degradation of Radix Isatidis Polypeptide in Artificial Gastrointestinal Environment

2014 ◽  
Vol 989-994 ◽  
pp. 1020-1024
Author(s):  
Nan Nan ◽  
Xi Jing Liu
Keyword(s):  
Gel Electrophoresis ◽  
Free Amino ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Radix Isatidis ◽  
Sds Page ◽  
Electrophoresis Method ◽  
Amino Acid Levels ◽  
Different Molecular Weight

Radix Isatidis is a traditional Chinese medicine for treatment of influenza and inflammation in China. In this paper, in order to study the degradation situation of Radix Isatidis polypeptide in artificial gastrointestinal environment, the SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method was used to detect the degradation of Radix Isatidis polypeptide in artificial intestinal juice and gastric juice, and it showed that Radix Isatidis peptides could be degradated to different degrees. HPLC (High Performance Liquid Chromatography) was used to determine the change of peptides degradation, and it indicated that free amino acid levels did not change significantly. The result after degradation was also detected by BCA method, and it showed that there were still a large number of polypeptides in the liquid. From this experiment we can come to this conclusion that Radix Isatidis polypeptides in artificial gastrointestinal juice mostly degraded into a series of different molecular weight peptides.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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